The present invention generally relates to a method to regulate airway hyperresponsiveness by modulating the action of xcex3xcex4 T cells in a patient. The present invention further relates to methods for identifying compounds that regulate airway hyperresponsiveness by modulating xcex3xcex4 T cell action.
Diseases involving inflammation are characterized by the influx of certain cell types and mediators, the presence of which can lead to tissue damage and sometimes death. Diseases involving inflammation are particularly harmful when they afflict the respiratory system, resulting in obstructed breathing, hypoxemia, hyperapnia and lung tissue damage. Obstructive diseases of the airways are characterized by airflow limitation (i.e., airflow obstruction or narrowing) due to constriction of airway smooth muscle, edema and hypersecretion of mucus leading to increased work in breathing, dyspnea, hypoxemia and hypercapnia.
A variety of inflammatory agents can provoke airflow limitation including allergens, cold air, exercise, infections and air pollution. In particular, allergens and other agents in allergic or sensitized mammals (i.e., antigens and haptens) cause the release of inflammatory mediators that recruit cells involved in inflammation. Such cells include lymphocytes, eosinophils, mast cells, basophils, neutrophils, macrophages, monocytes, fibroblasts and platelets. Inflammation results in airway hyperresponsiveness (AHR). A variety of studies have linked the degree, severity and timing of the inflammatory process with the degree of airway hyperresponsiveness. Thus, a common consequence of inflammation is airway hyperresponsiveness.
Currently, therapy for treatment of inflammatory diseases involving AHR, such as moderate to severe asthma and chronic obstructive pulmonary disease, predominantly involves the use of glucocorticosteroids and other anti-inflammatory agents. These agents, however, have the potential of serious side effect, including, but not limited to, increased susceptibility to infection, liver toxicity, drug-induced lung disease, and bone marrow suppression. Thus, such drugs are limited in their clinical use for the treatment of lung diseases associated with airway hyperresponsiveness. The use of anti-inflammatory and symptomatic relief reagents is a serious problem because of their side effects or their failure to attack the underlying cause of an inflammatory response. There is a continuing requirement for less harmful and more effective reagents for treating inflammation. Thus, there remains a need for processes using reagents with lower side effect profiles, less toxicity and more specificity for the underlying cause of AHR.
Airway hyperresponsiveness (AHR) is the result of complex pathophysiological changes in the airway. A variety of studies have linked the degree, severity and timing of the inflammatory process with the degree of airway hyperresponsiveness. However, the mechanisms leading to AHR are still poorly understood and can be attributed to both immune-dependent and immune-independent mechanisms. Essentially all of the T cell-mediated effects described so far are in the former category. However, T cells from hyperresponsive mice can increase baseline airway tone in hyporesponsive mice after cell transfer. Because of their constitutive presence in the normal lung, xcex3xcex4 T cells have been investigated with regard to their potential role in airway responses.
xcex3xcex4 T cells have been observed to proliferate and produce cytokines in many diseases. In addition, studies in animal models have provided evidence that these cells contribute to host resistance against infections (Hiromatsu et al., 1992, J. Exp. Med. 175:49), and that they can influence inflammation (Fu et al., 1994, J. Immunol. 153:3101), epithelial regeneration (Boismenu et al., 1994, Science 266:1253), and mucosal tolerance to antigens (Fujihashi et al., 1992, J. Exp. Med. 175:695; McMenamin et al., 1994, supra). Investigators are still determining what stimuli trigger xcex3xcex4 T cell reactivity, and to what extent xcex3xcex4 T cell activating stimuli differ from those of xcex1xcex2 T cells and B lymphocytes. It is known that xcex3xcex4 T cells respond during bacterial and viral infections, although they have not been readily linked to antigen-specific adaptive immunity.
A number of studies have investigated the presence and role of xcex3xcex4 T cells in diseases of the airways. Pawankar et al. noted the mucosal changes at the site of allergic inflammation in patients with perennial allergic rhinitis and chronic infective rhinitis includes an oligoclonal expansion and activation of Vxcex31/Vxcex41+T cells (Pawankar and Ra, 1996, J. Allergy Clin. Immunol. 98:S248-62). Molfino et al. showed that much of the xcex3xcex4 T cell population found in broncho alveolar lavage (BAL) fluid in humans derives from clonally expanded T cells (Molfino et al., 1996, Clin. Exp. Immunol. 104:144-153). Spinozzi et al., measuring xcex3xcex4 T cells in the BAL fluid from patients with asthma, concluded that allergen-specific, steroid-sensitive xcex3xcex4 T cells maybe one of the cellular components involved in the airway inflammation that characterizes allergic bronchial asthma (Spinozzi et al., 1996, Ann. Intern. Med. 124:223-227 and 1995, Mol. Med. 1:821-826).
Moreover, it has been noted that in patients with respiratory conditions including Bordetella pertussin infection (whooping cough) and asthma, circulating xcex3xcex4 T cells are decreased. It has been suggested that the reason for this decrease is the dispatch of xcex3xcex4 T cells to the site of inflammation in the lung. (Bertotto et al., 1997, Acta Paediatr. 86:114-115; Schauer et al., 1991, Clin. Exp. Immunol. 86:440-443; Krejsek et al., 1998, Allergy 53;73-77).
Many of the studies directed to xcex3xcex4 T cells and airway diseases have directly suggested that xcex3xcex4 T cells are proinflammatory, promoting acute airway sensitization, increases in cytokine levels suggested to be involved in allergic inflammation, regulation of allergic xcex1xcex2 T-cell and allergen specific B-cell responses, and/or allergen-induced eosinophilia and IgE responses (e.g., McMenamin et al., 1994, Science 265:1869-1871; Zuany-Amorim et al., 1998, supra; Schramm et al., 2000, Am. J. Respir. Cell Mol. Biol. 22:218-225; Schramm et al., 1999, International Conference of the American Thoracic Society; vol. 159:A255 (American Journal of Respiratory and Critical Care Medicine, San Diego, Calif.)). Some investigators, alternatively, have concluded that xcex3xcex4 T cells do not play a significant role in airway allergic inflammation. For example, Chen et al. noted, similar to other investigators discussed above, that allergic asthmatics have reduced xcex3xcex4 T cells in the peripheral blood. However, Chen et al. concluded that no significant correlation existed between the levels of xcex3xcex4 T cells and IgE present in the peripheral blood (Chen et al., 1996, Clin. Exp. Immunol. 26:295-302). Although allergic asthmatics have reduced xcex3xcex4 T cells with reciprocally elevated eosinophil numbers in the peripheral blood, Chen et al. asserted that this does not indicate that the reduction of xcex3xcex4 T cells correlates with the predominance of eosinophilia or IgE levels in diseased populations. Jaffar et al. described a role for xcex1xcex2, but not xcex3xcex4, T cells in allergen-induced Th2 cytokine production from asthmatic bronchial tissue (Jaffar et al., 1999, J. Immunol. 163:6283-6291). Fajac et al., 1997, Eur. Resp. J. 10:633-638 investigated the role of heat shock proteins and xcex3xcex4 T cells in patients with mild atopic asthma, and concluded that neither heat shock proteins nor xcex3xcex4 T cells play an important role in inflammatory and immune responses in mild asthma.
Therefore, prior to the present invention, those of skill in the art either considered xcex3xcex4 T cells to play an insignificant role, if any, in diseases of the airways, or believed that xcex3xcex4 T cells were proinflammatory cells which contributed to the development of acute airway hyperresponsiveness and other events associated with inflammation.
The present inventors have discovered that xcex3xcex4 cells can regulate airway function in an xcex1xcex2 T cell-independent manner, identifying them as important cells in pulmonary homeostasis. This function of xcex3xcex4 T cells differs from previously described immune-dependent mechanisms and may reflect their interaction with innate systems of host defense. Specifically, in contrast to other studies that emphasized their role in the modification of allergen-specific xcex1xcex2 T cell and B-cell responses, the present inventors have found that xcex3xcex4 T cells maintain normal airway responsiveness independently of xcex1xcex2 T cells.
One embodiment of the present invention relates to a method to reduce airway hyperresponsiveness in a mammal. The method includes the step of increasing xcex3xcex4 T cell action in a mammal that has, or is at risk of developing, a respiratory condition associated with airway hyperresponsiveness. In one aspect, the step of increasing xcex3xcex4 T cell action comprises increasing the number of xcex3xcex4 T cells in the lung tissue of the mammal. For example, the step of increasing can comprise removing xcex3xcex4 T cells from the mammal, inducing the xcex3xcex4 T cells to proliferate ex vivo to increase the number of the xcex3xcex4 T cells, and returning the xcex3xcex4 T cells to the lung tissue of the mammal. In another aspect, the step of increasing xcex3xcex4 T cell action comprises activating xcex3xcex4 T cells in the mammal. Activating xcex3xcex4 T cells can be performed ex vivo or in vivo.
In one embodiment of the method, the step of increasing xcex3xcex4 T cell action comprises administering an agent to the mammal that activates xcex3xcex4 T cells in the mammal. Such an agent can be any agent suitable for activating xcex3xcex4 T cells. In one aspect, the agent is a protein comprising a BiP-binding motif, wherein the protein is administered in an amount effective to induce proliferation of xcex3xcex4 T cells in the mammal. In another aspect, the agent is selected from the group consisting of a glycosylated protein and a glycosylated peptide. In another aspect, the agent is selected from the group consisting of polyGT and poly GAT (1:1:1). In yet another embodiment, the agent is selected from the group of: synthetic GC, synthetic AT and other oligonucleotides. In yet another aspect, the agent is a mycobacterial product. In another aspect, the agent is a Listeria cell wall product. In another aspect, the agent is a cardiolipin. In yet another aspect, the agent is tumor necrosis factor-xcex1 (TNF-xcex1). In one aspect, the agent is an antibody that specifically binds to a xcex3xcex4 T cell receptor and activates the xcex3xcex4 T cells. Preferably, the agent is an antibody that specifically binds to a xcex3xcex4 T cell receptor (TCR) from a xcex3xcex4 T cell subset that is particularly suitable for regulation of airway hyperresponsiveness. Such a TCR includes, but is not limited to, a murine TCR comprising Vxcex34 and a human TCR comprising Vxcex31.
In one aspect of the method of the present invention, the agent is targeted to xcex3xcex4 T cells in the mammal. Preferably, the agent is targeted to xcex3xcex4 T cells in the lung tissue of the mammal. In one embodiment, the agent is targeted to xcex3xcex4 T cell subsets that are particularly suitable for regulation of airway hyperresponsiveness, such xcex3xcex4 T cells having a T cell receptor (TCR) selected from: a murine TCR comprising Vxcex34 and a human TCR comprising Vxcex31. In one aspect, the agent comprises: (a) an antibody that specifically binds to a molecule on the cell surface of xcex3xcex4 T cells; and (b) a compound that activates the xcex3xcex4 T cells, wherein the compound is linked to the antibody of (a). The compound can include, but is not limited to: a protein comprising a peptide having a BiP-binding motif, a glycosylated protein or peptide, polyGT, polyGAT (1:1:1), synthetic GC, synthetic AT, a mycobacterial product, a Listeria cell wall product, cardiolipin, TNF-xcex1, and an antibody that specifically binds to a xcex3xcex4 T cell receptor and activates the receptor.
In one aspect of the present method, the agent is administered to the lung tissue of the mammal. In a preferred embodiment, the agent is administered by a route selected from the group consisting of inhaled, intratracheal and nasal routes. Preferably, the agent is administered to the animal in an amount effective to reduce airway hyperresponsiveness in the animal as compared to prior to administration of the agent. In one aspect, the agent is administered with a pharmaceutically acceptable excipient.
Preferably, the method of the present invention increases xcex3xcex4 T cell action within between about 1 hour and 6 days of an initial diagnosis of airway hyperresponsiveness in the mammal. In another embodiment, the xcex3xcex4 T cell action is increased within less than about 72 hours of an initial diagnosis of airway hyperresponsiveness in the mammal. In another embodiment, the xcex3xcex4 T cell action is increased prior to development of airway hyperresponsiveness in the mammal. Preferably, the step of increasing xcex3xcex4 T cell action decreases airway methacholine responsiveness in the mammal, and/or reduces airway hyperresponsiveness of the mammal such that the FEV1 value of the mammal is improved by at least about 5%. It is also preferred that the step of increasing xcex3xcex4 T cell action improves the mammal""s PC20methacholineFEV1 value such that the PC20methacholineFEV1 value obtained before the step of increasing xcex3xcex4 T cell action when the mammal is provoked with a first concentration of methacholine is substantially the same as the PC20methacholineFEV1 value obtained after increasing xcex3xcex4 T cell action when the mammal is provoked with double the amount of the first concentration of methacholine. Preferably, the first concentration of methacholine is between about 0.01 mg/ml and about 8 mg/ml. The method of the present invention is suitable for treating airway hyperresponsiveness associated with any condition including, but not limited to, airway hyperresponsiveness is associated with a disease selected from the group consisting of chronic obstructive disease of the airways and asthma.
Yet another embodiment of the present invention relates to a method to identify a compound that reduces or prevents airway hyperresponsiveness associated with inflammation. The method includes the steps of: (a) contacting a putative regulatory compound with a xcex3xcex4 T cell; (b) detecting whether the putative regulatory compound increases the action of the xcex3xcex4 T cell; and, (c) administering the putative regulatory compound to a non-human animal in which airway hyperresponsiveness can be induced, and identifying animals in which airway hyperresponsiveness is reduced or prevented as compared to in the absence of the putative regulatory compound. A putative regulatory compound that increases xcex3xcex4 T cell action and that reduces or prevents airway hyperresponsiveness in the non-human animal is indicated to be a compound for reducing or preventing hyperresponsiveness. Preferably, step (b) of detecting is selected from the group consisting of measurement proliferation of the xcex3xcex4 T cell, measurement of cytokine production by the xcex3xcex4 T cell, measurement of calcium mobilization in the xcex3xcex4 T cell, measurement of cytokine receptor expression by the xcex3xcex4 T cell, measurement of CD69 upregulation by the xcex3xcex4 T cell, measurement of upregulation of CD44 by the xcex3xcex4 T cell, and measurement of cytoskeletal reorganization by the xcex3xcex4 T cell.